A New Method for Mosaic Analysis of Clonal Mutations in Mice Abstract The advancement in genome technology has led to the discovery and accumulation of a large number of disease-associated genes. With the genetic information in hands, a bigger challenge and new opportunity now is to determine which genes or gene combinations can serve as indicators of health and/or potential targets for disease prevention and treatment. Mouse models provide the primary means to functionally verify genetic networks and causal links implicated from human genetic studies. With many recently developed technologies, such as CRISPR-CAS, it is possible to produce mutant mouse strains carrying mutations in any defined locus with significantly reduced cost and increased speed. Under physiological conditions most disease-associated mutations occur in single cells and expand clonally in somatic tissues of adult life. Studies suggested that somatic mutations and uneven clonal expansion contribute to many aging related human diseases, such as cancer, stem cell failure, and decline of immune functions. Whether a mutation is a driver or simply a passenger in clonal expansion and disease development is often inferred statistically or predicted by association studies, and only occasionally backed up with genetic validations. It becomes even a bigger challenge to investigate how multiple somatic mutations work together to promote disease development. Currently, the field still lacks robust tools to evaluate the causal links between compound somatic mutations and disease phenotypes. We have designed a new tool to induce and track clonal mutations in adult mice. When combined with any conditional alleles, this method can be used as a common tool to produce lineage- marked clonal mutations. This system offers three attractive features: 1) The generation of mutant clones is independent of mitosis and thus can be performed in any adult tissues, including quiescent cells. 2) It produces independently marked wild type and mutant clones at the same time in the same animals. The wild type clones can be used as internal controls for qualitative and quantitative assessment of the mutant phenotypes. 3) It can generate any combination of mutations within the same lineage marked clone. Thus, genetic interactions between different mutations can be tested within the lineage marked clones. The proposed study will further improve the design of this genetic tool to make it user friendly and with increased clonal frequency for mosaic analysis in the hematopoietic system. This newly established tool will be applied to clonal analysis of Id3 and Id2 mutations for their effects on the expansion, homeostatic maintenance, and transformation of hematopoietic cells. Both Id genes are important transcription regulators involved in lymphocyte development and lymphomagenesis. Outcomes of the proposed study will be a mosaic analysis tool with demonstrated utility in the study of immune cell homeostasis and tumorigenesis. The mosaic animal models can serve as either discovery tools for understanding gene functions, or as vehicles for developing and testing preventive or therapeutic regiments.